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I 


A 


THE  DETERMINATION  OF  SUCROSE  IN 
PLANT  EXTRACTS 


BY 


THOMAS  ELIJAH  HOLLINGSHEAD 


THESIS 

FOR  THE 

DEGREE  OF  BACHELOR  OF  SCIENCE 

IN 

CHEMISTRY 


COLLEGE  OF  LIBERAL  ARTS  AND  SCIENCES 

UNIVERSITY  OF  ILLINOIS 


1922 


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TABLIi  OF  GONTMTS 

Page 

Acknowledgement 

Nature  and  Origin  of  the  problem 

1 

Experimental 

Preparation  of  Pure  Sucrose 

9 

Purification  of  Glucose 

9 

Source  of  Asparagin 

11 

Comparison  of  the  Defren's, 
Ilunson- Walker , and  Clerget 
Methods  of  Sucrose  Deter- 

mina tkon. 

12 

Defren's  Method 

12 

Lfims on- Walker  Method 

13 

Clerget  Method 

13 

Experiment  I 

14 

Determination  Copper 

16 

Experiment  II 

17 

Experiment  III 

20 

Conclusion 

21 

Summary 

21 

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NATURE  AlID  QRIGII^  OE  THE  EKOBLSM 


The  v;ide  variation  hetween  the  sucrose  values  obtained  by  the 
plariscopic  and  chemical  methods  is  a source  of  difficulty  encount- 
ered by  quite  a number  of  workers  in  plant  chemistry.  This  has 
been  especially  true  v;hen  an  effort  was  made  to  determine  the 
quantity  of  specific  sugars,  as  sucrose,  glucose  and  fructose. 
Davis^  and  his  co-workers  found  differences  of  as  much  as  eighty 
per  cent  in  the  copper  reduction  and  polariscopic  values  for 

glucose  and  fructose  in  the  mangold  and  potato;  the  polariscopic 

2 

value  being  the  higher.  Parkin  also  found  the  polariscopic 
value  to  be  higher  than  the  chemical  value  in  his  work  v;ith  the 
carbohydrates  of  the  snow  drop.  Chemists  working  with  quite  a 
variety  of  plant  extracts  and  under  different  conditions  have 
reported  similar  results.^*  ^ 

Of  all  the  sugars  present  in  plant  extracts  it  should  seem 
that  sucrose  might  be  determined  most  accurately,  since  it  is  a 
non- reducing  sugar  and  on  hydrolysis  in  converted  into  glucose 
and  fructose,  both  of  v/hich  reduce  Fehling's*^  solution  which 
offers  a chemical  method  involving  the  determination  of  the  increas( 
in  reducing  sugar  after  hydrolysis.  The  fall  in  rotation  after 
hydrolysis  offers  a method  for  the  determination  of  sucrose  which 

7 

has  been  utilized  in  the  method  of  Glerget 

1.  Jour.  Ag.  Science,  _7,  349  (1915) 

2.  Biochem.  Jour.,  _6,  18  (1911-12) 

3.  Brown  and  Morris,  j.  Ghem.  Soc. , j^,  664  (1893) 

4.  Huncie  and  Englis,  Thesis  ( ) U.  of  Illinois 

5.  Browne,  J.  Am.  Ghem.  Soc.  451  (1906) 

6.  Bro;vn  "Sugar  Analysis”  p.335,  p.  389 

7.  Ibid  p.  264 


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But  as  mentioned  above  the  two  methods  do  not  check  in  the  case 
of  plant  extracts.  The  following  is  a typical  analyses  of  leaf 

4 

extracts  hy  Lluncie  • 

_ Sucrose  in  IJillegrams 

By  Polarization  Method  99.2  139.2  107.2  97.7 

By  Reduction  Method  94.3  132.3  101.9  98.8 

0 

Davis  assumes  that  the  difference  F;as  caused  hy  certain 

optically  active  nitrogenous  impurities  such  as  glutamine  and 

asparagin  which  are  present,  and  are  not  prepititated  hy  basic 

SI 

lead  acetate.  Somers  ^ and  latter  ^uisumhing  and  Thomas 
thought  tliat  the  auto-reduction  of  the  Pehling’s  solution  might 
introduce  an  error  large  enough  to  account  for  the  difference 
in  the  results  of  the  two  methods,  hut  the  data  from  their 
experiments  shov/s  that  this  error  is  quite  insignificant.  Somers 
also  stadiecT  the  effect  of  neutral  salts,  such  as  sodium  acetate 
and  oxalate,  upon  the  rotation  of  the  invert  sugar,  hut  found 
them  to  have  no  effect.  The  presence  and  effect  of  such  impur- 
ities as  Glutamine  and  asparigin  which  are  present  in  plant 
extracts  has  been  noted  hy  Pellet^,  Saillard^^,  Ogilvie  and 
others.  Ehrlich^^  pointed  out  quite  early  the  presence  of 
large  errors  in  the  Clerget  method  due  to  the  presence  of  amino 
acid.  The  error  that  he  refered  to  was  due,  however,  chiefly 
to  the  fact  that  amino  compounds  such  as  aparagine,  aspartic 
acid,  glutaminic  acid,  leucine,  isoleucine,  and  so  forth,  vary 

7.  Browne  "Sugar  Analysis  p.  264  9.  j^t.  Sugar  J.,  17,  421(1915) 

8.  J.  Agr.  3ci.,  ^,339,  (1916)  10.  Comp,  r end. ~Tb 5 ,116 

20.  Somers  and  Englis,  Thesis,  19 2()  ll.  Int.  Sug.  14,  89 

21.  J.A.C.S.  43,  1503-1526  12.  Z.  deut.  Zuckerind.53,809 


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in  optical  activity  depending  upon  the  alkalinity  and  acidity  of 

the  solution.  This  may  he  seen  from  the  following  table  v/hich 

gives  the  approximate  specific  rotations  of  several  amino  deriv- 

13 

atives  in  alkaline  solution,  in  v/ater  and  hydrochloric  acid. 

In  presence  of  In  presence  of 


HaOH 

In  water 

HCl 

Asparagin 

-8 

-6 

34 

Aspartic  acid 

-9 

4 

34 

Glutaminic  acid 

-68 

10 

20 

Leucine 

7 

— 

17 

Isoleucine 

11 

10 

37 

To  overcome  this  error  due  to  change  in  alkalinity  and 

acidily  of  the  solution  it  is  only  necessary  to  carry  out  the 

hydrolysis  by  means  of  invertase,  riBking  both  readings  with 

the  solution  neutral.  Or,  as  suggested  by  Andrlik  and  Stanet^^ 

both  polarizations  may  be  carried  out  in  the  presence  of 

hydrochloric  acid  and  urea,  depending  upon  the  retarding 

influence  of  the  urea  f or  betaine)  on  the  inversion  of  sucrose 

15 

with  hydrochloric  acid  in  the  cold.  Incidently,  Browne  offers 
data  to  prove  that  the  retarding  action  of  the  urea  is  not 
sufficient  in  the  case  of  substances  rich  in  sucrose.  If  there 
is  no  combination  or  reaction  between  the  sucrose  and  amino 
compounds  the  error,  under  the  conditions  stated  above  should 
be  constant,  and  not  effect  the  difference  in  the  direct  and 

13.  Browne  "Sugar  Analysis"  p.  270 

14.  Ibid,  p.  271 

Z.  Suckerind.  Bohmen,  417 

15.  Bro;vn  "Sugar  Analysis",  p.  271 


-4- 


and  invert  polarizations*  This  may  perhaps  be  illustrated  by 
an  equation: 

D I = d 
(Dia)  - ( I±a)=  d 

in  which  D equals  the  direct  polarization,  I the  invert  polar- 
ization, a the  polarization  of  the  amino  compound  which  is  a 
constant,  and  d the  difference  in  the  tv/o  polarizations.  Unless 
there  is  some  reaction  between  the  sugars  and  the  amino  compounds 
thise  equations  should  hold  true,  and  the  presence  of  amino 
derivatives  should  not  be  the  cause  of  difference  in  the  values 
obtained  by  the  polarimetric  and  chemical  methods  as  suggested 

Q 

by  Davis  and  his  co-workers  in  their  work  on  the  determination 

of  the  glucose-fiructose  ratio  in  mangolds* 

15 

Degener  , a French  chemist,  found  that  asparagine  may 
introduce  an  error  in  the  determination  of  sucrose  in  sugar 
beet  extract  by  plarization*  He  has  shown  that  asparagin, 
v;hich  in  neutral  solution  is  ^ ightly  levo-rotatory 
becomses  strongly  dextrorotatory  (Cf3  j;)  5 61.76  to  69.10t  in  the 
presence  of  10  per  cent  lead  subacetate  solution,  every  0.1 
per  cent  asparagin  polarizing  about  the  same  as  every  0*1  per 
cent  sucrose*  To  obviate  this  error  the  French  chemists  add 
a drop  of  glacial  acetic  acid  to  the  filtered  sucrose  solution 
before  polarizing* 

As  stated  above, none  of  these  considerations  account  for 
the  very  large  variations  between  the  polariscopic  and  copper 


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reduction  methods.  There  is  very  evidently  some  greater  source 
of  error,  which  may  he  due  to  some  unstable  compound  formed  by 
the  sugar  and  the  amino  substances,  or  possibilly  to  some 
catalytic  effect  upon  the  reducing  power  of  the  glucose  and 
fructose  formed  by  the  hydrolysis  of  the  sucrose. 

Thacher  says  no  nitrogenous  groups  of  the  protein  type 
have  been  found  in  combination  v/ith  sugars  in  glucosides. 
Browne-^'  mentions  some  animal  substances,  which  are  not  of  a 
pure  carbonhydrate  nature,  yielding  glucose  as  one  of  their 
hydrolytic  products.  Among  these  substances  may  be  mentioned 
different  nucleo-proteids , various  albumins  and  certain  mucins 
or  mucoids.  The  chemistry  of  these  products,  hov/ever,  is  still 
unsettled,  and  it  is  uncertain  whether  the  sugar  derived  from 

consists  of  glucose  alone  or  a mixture  of  sugars. 

22 

Somers  describes  an.  experiment  to  stady  the  effect  of 
asparagin  on  the  polarization  of  invert  sugar,  which  is  as  fol- 
lows: "The  asparagin  was  from  a laboratory  stock  solution  and 

the  apparatus  used  was  a Schmidt  and  Haensch  polariscope, 
a half-shaded  instrument,  calibrated  in  Ventske  degrees.  The 
asparagin  and  invert  sugar  solutions  were  mixed  and  polarized 
in  accordance  with  the  following  table 


A 

B 

G 

Asparagin 

25  c.c. 

25  c.c. 

00 

Invert  Sugar 

00 

25 

25 

kVa  ter 

25 

00 

25 

Polarization  Reading 

-.320 

1.438 

-1.060 

16.  Thatcher  "Chemistry  of  Plant  Life"  , p.76 

17.  Browne  "Sugar  Analysis"  p.S79 
22.  Somers  and  Englis,  Thesis,  1920) 


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-7- 


The  mixed  asparagin  and  invert  sugar  solution  give  a 
greater  negative  rotatory  value  than  that  obtained  by 
adding  the  rotation  values  for  the  constituents  determined 
separately.  This  seems  to  indicate  that  there  is  some  sort 
of  loose  combination  between  the  sugar  and  asparagin,  and 
it  seems  plausible  that  it  may  be  responsible  for  at  least 
part  of  the  disparity  between  the  tv/o  methods  for  the  est- 
imation of  sucrose.  He  also  made  an  attempt  to  repeat  the 
experiment  v/ith  a neutral  solution  of  glutamic  acid  hydro- 
chloride but  there  was  such  a development  of  color  that  it 
was  impossible  to  determine  the  polarization  value  of  the 
solution. 

Stanek^®  found  that  when  he  heated  solutions  of  sucrose, 
or  invert  sugar  with  sodium  glutamate  or  aspartate  or  v/ith 
asparagin  at  a temperature  of  one  hundred- five  to  one 
hundred- thirty  degrees  Centigrade  that  carbon  dioxide  was 
liberated,  the  solution  became  acidic,  and  dark  pigments  were 
formed  which  are  almost  completely  precipitated  with  lead 
acetate.  Asparagin  and  aspartic  acid  being  the  most  reactive. 

Kaillard  found  that  there  was  an  actual  reaction 
between  glucose  and  glycocoll  when  they  were  dissolved  in 
three  or  parts  of  water  and  warmed.  The  liquid  slov/ly 
assumed  a characteristic  yellow  color,  changing  to  a dark 

18.  J.  Chem.  Soc.,  112.  I,  544 


19.  Comp.  rend.  154 , 66 


rf 


sa.'UMet; 


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brown,  follov/ed  by  the  evolution  of  carbon  dioxide.  It  has  been 
found  that  the  carbon  dioxide  came  from  the  carboxyl  group  of 
the  glycocoll.  It  v/as  assumed  that  this  loss  of  carbon  dioxide 
is  accompanied  by  an  union  of  the  nitrogen  v;ith  the  aldehyde  carbo: 
of  the  sugar;  the  glucose  molecules  forming  part  of  the  new 
compounds  must  suffer  dehydration  resulting  in  the  appearance  of 
double  bonds  or  possibly  rings.  It  v/as  also  shovm  that  other 
amino  acids  work  in  a similar  if  not  identical  manner  on  glucose, 
and  other  sugars.  This  seems  to  prove  that  sugars  and  amino 
compounds  can  react  under  certain  conditions  to  form  new  compounds 
hence  it  seems  plausible  that  they  might  have  some  effect  upon  one 
another  under  ordinary  pressure  and  room  temperature. 

The  object  of  this  investigation  is  to  study  the  effect  of 
asparagin  upon  the  rotatory  power  and  reducing  power  of  sugar 
solutions. 


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-9- 


EKPEHIIvIMTAL 

Preparation  of  Pure  Sucrose  ^ *2,5 

Method  of  the  International  Commission  for  the  Unification 
of  Sugar  Analysis* 

A hot  saturated  aqueous  solution  is  prepared  end  the  sugar 

4 

is  precipitated  v/ith  absolute  ethyl  alcohol  ; the  sugar  is 
carefully  spun  in  a small  centrifugal  machine  and  washed  in  the 
latter  with  absolute  alcohol.  The  sugar  thus  obtained  is  re- 
dissolved in  water,  the  saturated  solution  again  precipitated 
v/ith  absolute  alcohol  and  washed  as  above.  The  product  of  the 
second  crop  of  cry stalls  is  dried  between  blotting  paper  and 
preserved  in  glass  vessels  for  use.  The  miliisture  still  contained 
in  the  sugar  is  determined  and  taken  into  account  when  weighing 
the  sugar  which  is  to  be  used. 

Sucrose  prepared  by  the  above  method  was  found  to  contain 
only  0.05%  moisture  and  about  0.1%  ash. 

Purification  of  Olucose 

The  raw  material  is  the  commercial  com  sugar,  "Gerelose", 
manufactured  by  the  Corn  Products  Refining  Company,  of  New  York. 

Five  hundred  grams  of  the  dry  corn  sugar  are  dissolved  in 
2.5  liters  of  cold  water,  about  20-30  grams  of  decolorizing 
carbon,  such  as  "eponite”  or  "norit”,  are  added  and  the  mixture 
is  stirred  for  a few  minutes. 

1.  U.S.Dept.  Agri. ,Bur.Ghem.  Bull.  73,  p.58 

2.  J.A.M.G.  59 

3.  Sherman,  Methods  of  Organic  Analysis,  p.91 
5.  Ibid,  p.97 

4.  vV. A. Noyes,  Organic  Chem.  for  Laboratory,  pp.  67-68 


r 


-10- 


It  is  then  heated  to  about  97®  C.,  and  niade  just  acid  to  litmus 
with  a few  drops  of  phosphoric  acid  (H^PO^)  and  filtered  through 
an  asbestos  mat.  The  colorless  filtrate  is  evaporated  in  a glass 
flask  under  reduced  pressure  to  a content  of  70  to  75^  solids. 
Glacial  acetic  acid  is  now  added  in  the  ratio  of  one  part  of 
acid  to  one  part  of  sirup.  This  Solution  is  placed  in  a jar, 
seeded  with  pure  glucose  crystals,  and  stirred  occasionally. 
Crystallization  v;ill  commence  almost  immediately,  v/hile  the  sol- 
uition  is  still  warm.  It  is  well  to  stir  in  one  more  part  of 
acid  as  cry s tall iz.at ion  proceeds  to  prevent  the  formation  of  a 
solid  mass  of  crystals.  Crystallization  is  complete  in  a few 
hours  or  over  night.  The  thick  mass  of  crystals  is  then  filtered 
from  the  mother  liquor  on  a Buchner  funnel  fitted  with  a strong 
filter  paper,  and  v/ashed  with  glacial  acetic  acid.  After  draining 
as  dry  as  possible  on  the  Buchner  funnel,  the  crystals  are  washed 
v/ith  95^0  alcohol  and  dried  in  a vacuum  oven  at  50®  G.,  this 
temperature  may  be  raised  to  70°  C.,  in  the  course  of  one  or 
two  hours . 

Tv;o  runs  were  made  using  this  method.  In  the  first  the 
yield  was  very  low  due  to  insufficient  concentration  of  the 
sirup  before  crys tallization.  The  Abbe®  refractometer  may  be 
used  to  advantage  in  estimating  the  total  solids  of  the  sirup. 

The  product  of  the  first  lun  was  not  analyzed.  In  the  second 
run  about  350  grams  of  dry  pure  glucose  viexe  obtained,  which 
upon  analysis  shov/ed  0.065  per  cent  moisture,  end  ash  0,199  fo, 

8.  Browne  "Sugar  Analysis”  pp.  50-75. 


,',eM:"i:i  ♦♦0'  ':«,i:.  ,^I 

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mm  . ■ ^'  v:/_.  ^ > ,f  ., 


-11- 


The  product  is  colorless  and  its  solution  is  colorless  and 

free  from  cloudiness*  It  is  the  anhydride,  and  is  ^nixture  of 

the  alpha  and  beta  d-glucoses.  The  method  works  v;ell,  and 

practically  the  only  difficulty  encountered  v/as  the  absolute 

9 

removal  of  all  the  acetic  acid. 

The  Com  Products  Company  are  noMJ  putting  on  the  market 
a purified  glucose  which  is  crystallized  from  v/ater,  therefore 
free  from  any  acetic  acid.  The  method  they  use  is  described 
by  Porst  and  Humford  in  the  Llarch  Journal  of  Industrial  and 
Engineering  Chemistry,  Vol.  14,  No. 3,  (1922). 

Source  of  Asparagin 

The  asparagin  used  in  this  work  was  obtained  from  Herk 
Company.  It  was  the  1-asparagin  v/hich  has  the  following 
formula  and  properties : 

C-  OH 

I 

CE-  HH„ 

VO 

C - HHg 

Asparagin  or  Amido- amino- succine 
A ten  percent  HCl  solution  of  asparagin,  with  the  sodium 
line,  gives  a rotation  of  37.27?  a neutral  solution  -6.14°; 
and  16.4  grams  in  100  c.c.  ammonia  solution  gives  a rotation 
of  -271°. 

The  material  obtained  from  Merk  in  water  solution  gave  a 

rotation  of  aproximately  -6°. 

9.  Hudson  and  Dale,  J.A.G.S.  320 
10.  Abderhalden,  Biochem.  Eandlexikon,  Yol.4,  p597 


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-12- 


Asparagine  is  only  slightly  soluble  in  water  at  ordinary  temperat- 
ures. 


20.5° 

G. 

100 

parts 

of 

water 

will 

dissolve  0.62 

P* 

Asparg 

31.50 

» I 

1 I 

1 1 

1 1 

1 I 

I 1 

" 0.75 

1 1 

» 1 

46° 

I t 

1 t 

1 1 

1 1 

1 1 

I 1 

” 1.14 

1 » 

1 1 

70° 

1 I 

1 1 

1 1 

1 1 

1 1 

1 1 

” 2.25 

1 I 

1 1 

It  is  considerably  more  soluble  in  acid  or  alkaline  solution. 

Comparison  of  the  lefrens « llunson- V/alker , and  Qler^;et 
Methods  of  Sucrose  Determination. 

Defren's  Method 

In  Defren’s  method,  which  adapted  from  O’Sullivan,  Soxhlet’s 

12 

formula  for  Pehling’s  solution  is  used:  15  c.c.  of  the  copper 
sulfate  solution  and  15  c.c.  of  the  alkaline  tartrate  solution  are 
diluted  with  50  c.c.  of  water  in  a 300- c.c.  Erlenmeyer  flask. 

The  latter  is  then  immersed  for  5 minutes  in  a boiling  water  bath, 
w'hen  25  c.c.  of  the  sugar  solution  are  quickly  run  in  from  a 
burette  or  pipette.  The  flask  is  replaced  in  the  bath  and  heated 
for  exactly  15  minutes.  The  cuprous  oxide  is  then  filtered  on 
asbestos,  washed  with  warm  water,  ignited  and  weighed  as  cupric 
oxide,  or  it  may  be  dissolved  in  nitric  acid  and  determined  vol- 
ume trically.  The  amounts  of  glucose,  maltose,  lactose  and  inveit 

sugar  corresponding  to  different  v/eights  of  cupric  oxide  are  given 
in  tables. 

11.  Brov/ne,  "Sugar  Analysis"  p. 425-6;  J.A.G.S.  18,  751 

12.  Ibid,  389-90, 


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-■■  « ■ K ' r: 

■.  ,i'.'»4'.1  '.1"  tri  tl  V ;.,.  ,*(>1  .,  "tS  #t/*’‘'S-'u  . . 

ifir  ■ ■ ‘.  ' • ',  N/fi  t ' 4'  . ' 


H 


',  r'  ■ ' , • *\  . . »'  ' \ • ^ 


.r^b  , 

; f 

:i  ■m'<ii 


( tJ^  » ?*  * W • » A t 'vf  -1,^1  -*\\ 


■ ■ ''■;  ■' ^.;:V':,  .,'  . > ti 


i 


...•( 


-13- 


Ltimson-V/alker  llriified  Ivlethod^^ 

Transfer  E5  c.c.  each  of  the  copper  and  allcaline- tartrate 
solutions  (Soxhlet's  formula)  to  a 400-c,c.  Jena  or  Non- sol 
beaker  and  add  50  c.c.  of  reducing  stigar  solution,  or,  if  smaller 
volume  of  sugar  solution  be  used  adA  water  to  make  the  final 
volume  100  c.c.  Heat  the  beaker  upon  an  asbestos  gauze  over 
a Bunsen  burner;  so  regulated  that  boiling  begins  in  4 minutes, 
and  continue  the  boiling  for  exactly  2 minutes*  Keep  the  beaker 
covered  with  a watch  glass  throughout  the  entire  time  of  heating. 
V/ithout  diluting  filter  the  cuprous  oxide  at  once  on  an  asbestos 
felt  in  a porcelain  Gooch  crucible,  using  suction.  V/ash  the 
cuprous  oxide  thorougly  v;ith  water  at  a temperature  of  about 
60®  C.,  then  with  10  c.c.  alcohol  and  finally  with  10  c.c.  of 
ether.  Dry  for  30  minutes  in  a v;ater  oven  at  100®  G.,  cool  in 
a desiccator  and  v/eigh  as  cuprous  oxide.  Tlie  amiounts  of  glucose, 
invert  sugar,  lactose  or  maltose  corresponding  to  different 
v/eights  of  cuprous  oxide  or  copper  are  given  in  tables. 

Qlerget  Method 

Dissolve  26  grarasoof  the  sugar  in  60  c.c.  of  water,  clarify 
if  necessary,  dilute  to  100  c.c.,  filter,  and  polarize  at  20°  0. 

The  solution  is  then  deleaded  by  means  of  anhydrous  sodium  or 
ammonium  carbonate,  or  disodium  phosphate^^,  followed  by  filtrat- 
ion. To  50  c.c.  of  this  solution  25  c.c.  of  v/ater  are  added, 
then  5 c.c.  of  cone.  HGl  with  constant  shaking,  and  the  solution 
is  made  up  to  100  c.c.  at  20®  Q.,  and  allowed  to  stand  over  night 

13.  Brown  "Sugar  Analysis"  p.426  15.  Sherman "Organic  Analysis"p.94 

14.  Ibid,  264-286 

16.  Englis  and  Tsang,  J.A.G.S.  M,  865  (1922) 


U-t.*]’:.  tK'-  H‘ 


X) , 5 A "ij  ;>-  ^ 


j -.  fms^  ^ii  * • , v/- ,i  ^ 

} ■ 4.‘..  ' ,..;  '..>  ^.v  ^ -^(VA  nfwlfiru^ji  < 

^ , L&  .•  - ' ;_.£_  '■  ^^‘•vS> 

^<1  -tL  _^lJL^|wjjj^J  '*■*'*  ‘*0;^’^'^  ' TEKii- <’' < tV'*  ? | - 


V i ♦: 


■'  V. 


“'  ’.'‘T-if»A  v|w  . :i'.«  CP'  V>  V .1  cc;'.;j4f*:4?  o:;^t’W^l^'I!W  it 


'i.i^ 


• f. 


t 


;*i.f » 


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* ' '■  ■•'’  ’ •'  'S}’^  '■'  f“  ''  . 7 'V  ''J>' 

; ■:.  v..'‘  -.-ii  ' 


- » 7'  - y-j 

■ r .-.r-OX  «i}-^ 


\n  ••» 


M-' 


^ • •V*0  tu,Ui,':>‘ 


t . 


. «5J-  9^'X  -tif  ntt-Ov  • 

I *i’’V.  ' .>  '//•  ,"' '.'’'  •■  ‘•. , '.  ■'  .•?.  ..  ^ i> • ^r.’*{l’}i 


•pi-^ 


■"■^  v"  u ■"  *=  " w*  . » >"••:  ' • -A-.:  ■•vtMB  ' 

' ' •„  V :>  ,:.^j.^  ■ ’ , 


\ •' 


’ '1  i'  L4  ^ ' * 

V . A Wi  • »f  ‘,H  • 


r. 


.._  . 

♦istSI.i'i  Agp'i^' 

fY-:i  ^ ^ ; i 

V'  iV;  p ‘Mr  1 

■ -:;■  -r-’p-  ■•y'^Ar':^  .^  ■ Sr  ' 

wirli  4«'  .*•■€(;. or  ■ ^7  * v - ^.i  • ; 'jL 


I 


t ' 


••■•1  l..n^  r tv  . fcftuMi  U vty^ 

ipTli..  ’ „ . ■;  7■.<^^4.T‘V^4*H^  . . V 4 V,  _ *.  wwi 

^ ^ ‘ ^1?'  ■ - 1 MJ'  : ^ ' 'uiP\  '■■''  ■ ^ 


-14- 


at  between  25^  and  30°  Centigrade.  Or  the  hydrolysis  may  be 

17 

carried  out  by  use  of  Inver tase  in  stead  of  the  acid,  which 
tends  to  decompose  some  of  the  fructose.  Cool  the  solution 
to  20°  C. , mix  the  solution  well  and  polarize,  preferably  in  a 
waterjacted  200  -mm.  tuve  with  a wide  tube  through  which  the  exact 
tempeature  of  the  solution  at  the  time  of  polarization  may  be 
determined.  Since  50  c.c.  of  the  sugar  solution  v/as  diluted  for 
hydrolysis  and  finally  made  up  to  100  c.c.  this  second  reading 
must  be  doubled  to  obtain  the  "invert  polarization".  The 
difference  between  the  first  plarization  and  the  "invert  polar- 
ization" multiplied  by  100  and  divided  by  the  difference  which 
pure  sucrose  would  show  under  the  same  conditions,  gives  the 
percentage  of  sucrose;  or 

s > 100  {P  - I) 

142.66  - 0.5t 

in  which  s equals  percentage  of  sucrose,  P the  original  polar- 
ization, I the  "invert  polarization",  and  t the  temperature  in 
degrees  centigrate. 

■Experiment  I 

A sample  containg  12.5  grams  of  pure  glucose  was  analyzed 
by  the  Defren’s  , LIunson  -Walker,  and  the  polariscopic  method.  > 


17.  Sherman,  J.A.C.S.  1566 


- .'Tain*  xt:.- 

V 

IWHPIP  ' '■  ’ ’ ■ . 

'"ui  ■ - !l a •*'^'''’-^'V>?r*’-'||i  ” ^»v' 

'\  


i ■- ’if’ f!  , .;  t u >c  •'<  ^ v'ti  Jf : :l%xa©  gjn 

^’  ■ 1'  ' • ' ' F ..  ' .'  i 

*-v,i.i  ..:u^|  OS'..  XOqO  ‘avt  ’*^  ».  ■ 


a {.' 


: »■’■■. 


',  •■> j . . , ^ ' '1  - ■ ^*1, 

:.>•,  . , j!  / .:»v)kV*,  .ti 


; ai^ 


-t  ^ ^ 

, » W « • ^ .1 


< ~ t 

Uv  >*«41iFt.  .:  ’>  . 'CtTVjU" 


Vtot  u n< 

\ 


• -I  - < ■ 


t-r*aw(Pai|r:ur  . v'.  . -Vi:  v>’ q;.  iriiifin  :fe£S> 

^ ^ ^ r*r"  ' ^ ■■  V.  -'  - ^ ' ^ ‘ ^ ‘.  » ^ 

5Li/,- . '■-■>  ■ ■■••'.ii  • *‘”'  ' Svj 

■.  :Vwaj^ . ■-.•  .a**^  ^ i)i(  ..^ ‘ ' 

■-  ^■‘'\-ji  *■  M '.A,  , 

^2“  :;.t  ^ B OflP  fit- ,M4  r<? 


z,'.t  .j^.  -f  t-a  (7  .1/;,1  V'  :,^ii»>'.  M(.t^  ■'  r>4(i 

I'  , ««r.  J .’  .,  . ,T  '-,,  ,'  , ^ 


' ,t  >' 

1‘ 


*V  ■ 


‘i  r ,45'-«*  '■j  *J 


* * 


V ■ 

’■  , V^.  .V*  Aj^'r  ^ •',  ,,  ^ 

" **■  «o. >..%,;  .,,,;;* 


'i^.- 


•j  ^ r ' 


(\ffa  ,v  , 


:■  .U.,1; 


ff  j*. 


' ■ 4 


1 vi.'' 

j.  , -'/'V 

.,"  ■ m 

/f 

, > X k* 

;>’■ 

:.r-  -'t: .. 

’ ^ V>- 

\tk. 

:M 

m,  - 

i . .— 


k-'.> 


:i 


^ ^ ft  ' ■«  Jl 

. iW.  *i  4.  ■■  • ' , v|5  ,*,il?IL^ 

»;  4 '^"  ,’  ,'.  V*  ' '''i 

,;  ;..  ■ -^L 

t *r‘f^  I— ti^  > »ii -»■»■>  •#*»■  . kWi| 

«.^.  r - , •■  li  i’’'*'4|| 

- ' .rvf  \ V: 

' ill  'k  . ■'■  'V  ' -SI,  ' *«■ 


W^.  •"•  '''^'  •'■  ■ 


' .'(-3 


- - 1 
I*  * >•  •:<■  ■<  ^ 

'5  — ' ''V-, 

.'i^.duQR'  i‘  l^;«r  > ’'ii 


7T®*®JWWf 


I l^.;*!:  J '» 

k x kTM 

^ ,,  i4:t;1l 


-15- 


D ef r en ’ s Me  thod 

I 

II 

111 

C«C*  NagSgOg 

32.56 

32.52 

32.65 

Y/t,  of  CuO 

279.1 

279 

280 

’ * ’ ' Glucose 

125.61 

125.6 

126.1 

Per  cent  ’ ' 

12.561 

12.56 

12.61 

ifuns  on-  Walk  er  Me  th o d 

I 

II 

III 

C.C.  HagSgOg 

35.53 

34.1 

55.22 

Wt,  of  Gu 

242.5 

233.5 

241.0 

Wt.  ' ’ Glucose 

123.9 

119.0 

123.15 

per  cent  ^ ’ 

12.39 

11.90 

12.315 

Polariscopic  Method 

Reading 

37.7°  V. 

Per  cent  Glucose 

12.44^ 

Averages  and  Qomparison 

or  Results 

Defren's  Average-- 

- 12.577;^ 

Liunson-  Walker  ’ ’ 

- 12.202/5 

Polarimetric  

- 12.44  fo 

In  comparison  v\ith  the  polarimetric  results  the  Defren 
Method  shows  a difference  of  .137^  and  the  Munson- Walker 
Method  of *238  In  using  the  Munson- Walker  Method  a good 

deal  of  experience  is  required  to  tell  just  v/hen  boiling 
actually  starts  and  to  adjust  the  flame  to  start  the  boiling 
exactly  at  the  end  of  four  minutes.  This  difficulty  may 


, ^ ■,  ,'*■.  ^ it"'  ' . ' '' 


4rf-  » Xa1  ‘ ,' 


'a» 

j 


^ *»  - I 


■ 


aCOI 

. ■«  I • : 


•'■•V  Hi-iOh*'  i?X  t im.'lt  : 'Oilf.tt 

■'  ' ■ ''  ''  V E?.  . 


*'v  ^--  .ifna/5i 


ji  ■ ' r ®»'<  f’"?'.-'  1 

To  I^lit'  t'  t:'  ' 


>“■»•■  <»?  |^X|#r^B^^1CiB4! <» ; t.t- 1' •'UIT S j ^ ? 


W'.;  ^ 

V 


, l^/>-  ‘V 

* iu  ( : 


s3»'04  m 


yrorjA-**-! 


- i6- 

accoimt  for  in  part  the  poor  checks  obtained  by  the  I.fimson- Walker 
Me thod, 

4.  r.  18,  19,  20 
Determination  of  Copper 

The  copper  reduced  in  these  experiments  was  determined  by 
a modification  of  tlie  Low  lodometric  Method.  The  Cu.-0  is  dissolved 

Cj 

on  the  Gooch  by  pouring  over  it  10  c.c.  of  nearly  boiling  4N  HNO^. 

This  is  added  very  slov/ly  while  applying  mild  suckion  to  prevent 
loss  by  splashing.  Allov/  the  copper  nitrate  and  nitric  acid  to  be 
sucked  down  as  completely  as  possible,  then  wash  v/ith  five  or  six 
5 c.c.  portions  of  boiling  water.  Boil  for  a few  minutes  to 
remove  the  oxides  of  nitrogen,  add  5 c.c.  of  saturated  bromine 
water  and  boil  until  until  the  excess  bromine  is  expeSiled.  Remove 
the  flask  from  the  flame  and  add  strong  ammonia  until  a slight 
excess  is  present.  After  boiling  off  the  excess  ammonia,  add  10 
c.c.  of  30  fo  acetic  acid,  which  dissolves  any  copper  oxide  that  has 
been  deposited.  Cool  to  room  temperature,  add  3 grams  of  potassium 
iodide  and  tirate  the  brov;n  solution  with  standard  sodium  thiosulfat  . 
until  nearly  colorless,  addiiig  starch  solution  toward  the  last  and 
complete  the  tiration.  In  making  the  titration  for  the  first 
time  one  is  bothered  somevirhat  by  the  fact  fh&t  the  cuprous  iodide 
is  of  a light  brovmi  color.  This  difficulty  is  very  much  overcome 
by  keeping  dovm  the  amount  of  thiosulfate  solution  which  it  is 
necessary  to  use. 

In  standardizing^  the  sodium  thiosulfate  solution  a known  amount 

of  metallic  copper  is  used  to  begin  with  and  a regular  determination 
18.  Treadwell  Hall,  Vol.II,  pp.  682-683 

Browne  "Sugar  Analysis”  pp.411^13{ various  modification^  of  method) 

20.  Peters,  J.A.O.S.  422 


1 'V 

i - 

?■» 


. iV  ' ” 'i'-V'  '" 


•m 


*-4i  -V  ' '.  ’ 

v*<i  .‘'fom  •' 


t ‘ 


♦’.i 


» ^ »• 

r 


T y/S'i# . ■::  '1  ’.  mg 

jfjL  *'  ' # ‘ 


Vj‘  'i.t^  *.^0  ’>»5t  ;i>" 

. L^W  1 ' .1  ‘ , * ’ ■ ' ^ 1 

7i=>tJb^f.  til  -■  ,;uV  . Q#4rt»u-  ft  v'W>6(tf  > ,.'iJ>' 

J'  ' . ' -P-  '' -.'^j'lS  'in.^ 

^ . -?'4r*  <•  > "'j.  j 1 5j:.c^^»i  *■  ■“'  '.o*  :>  •«;  tf'i' 


u * -"- 1 i JJ  -■  ^ irrfv 


f lo^  i>H4^f:  h^‘i  .‘  l)  * . ,THi  S.OIXI 

* n ' . / * .mi 


‘^1 


• <Wit 't,  ‘'■^, -L^‘  ^ 


V'i  ■■ 


■VTtlr'f'^v  f'v.H 


. , . »,  _ C'^:t  ta  l0 ; 


N!^J  f ' > 


■4.  V: 


\ 


-pr  :■*•.•/■  .•  XjQU 


ifr., -‘Hr  .‘i^T  I- 


'•; ' -‘ra • 'yy'i'-iEI  '■.  .^,.‘*  • ' " V' 

'.1  V 6‘it  ;•  • 0l 


.rift  '71 


‘■^.  ; 


•P": 


)<»  *4^ac 

W ‘1 . r ,'  ■ 


■ l,v',^' 


V, 


'If  r,^ 

:j'  Ia'CjX  d,V 


r Aif.  , . - ;.  .■  |^.ciXC'a  N»£?!^6  <.  X V. 

tsJ  V iX  } ft  \i},s.  '.f|7,  ’ . rtf^  t'4  |\’i  * V isl^  f 

' -'\\4m  '■.*,  ■ ^’■  ■■ 


>iv*a<#'lv,  V ■:■  X'AW» 


i i - 

■ ' l «t« 


S' 


■>  • ■ 


t4  .T<iXoe  f:t.'04Dtr,  wttf  t:..' *»^C 

» ; • *1.  ■»..',  vv..‘ . .•  rr  "i^... 


roJrftT  4|..: '.  'i.i  .>.^*41^ 

«•■  . ,.  ■ ■•-  tiL  7.  ■ ,^,  .'  m‘'.<t; 

H . 7, ■":  'VJK?«  ^ o 


■^.m  ■/ 


•A' ^ • -4 «■'.;.  ij  Ui'.  '.A.'VI  t ftl 


■7' 

;•'  »V  , ^ ' . 


JLVI 


‘ liwt. 


\H‘ 

4 . 


Sn7 '/i' ,0i/;'cjjr;-;  '5  0^1  ^.1 

r ',  X jfiflff ' ixii’ t 

’’f  k^^av:  ■ 'iC •,(?^4 Iftt. 

^ V t . t-  ♦ ;.;,'.p ' , sn:©(i'»^y^r 

■■‘f  li-  ■'  V ■'  . 


1 ifc!' , i . ''  ' 


ft;  .'* 


Ti'l 


■i(.l  . a ' ..V  . I JK  H'l  Ml /‘4  ' ’ t'  )>  f •- 2 


1 


-17- 


is  carried  through.  Tlie  copper  value  of  the  solution  is 

calculated  by  dividing  the  grams  of  copper  used  by  the  number  o£ 
c.c.  of  solution  used. 


where  A is  grams  of  Copper  used,  t is  c.c.  of  used,  and 

C.V.  is  the  copper  value  of  each  c.c.  of  solution. 

With  a little  experience  just  as  good  checks  may  be  obtained 
by  this  method  as  v/ith  gravimetric  methods,  and  it  eliminates 
a very  great  amount  of  weighing  where  a great  many  samples  are 
being  run  at  the  same  time. 

The  only  difference  in  the  above  method  and  that  of  low  is 
that  in  the  above  method  10  c.c.  of  30^  acetic  acid  is  used 
instead  of  3.5  c.c.  of  glacial  acetic  acid,  as  3.5  c.c.  is  an 
a'/'kward  volume  to  handle. 

Experiment  II 

The  copper  reduced  by  solutions  of  invert  sugar,  glucose, 
sucrose,  asparagine  rn^v/ater  solution,  invert  sugar  in  presence 
of  asparagine,  glucose  in  the  presence  of  asparagin,  and  sucrose 
in  the  presence  of  asparagin  v;as  determined;  also  the  rotatory 
values  for  the  above  solutions. 


IjiPVIi 


-18- 


Invert  Sugar 

I 

II 

c • c • Q ^ , 0 

12.51 

»vt.  of  6u6 

11.8 

Invert  Su^ar  and  Asparafiin 

c.c.  NaoSpOg 

14.04 

14.04 

Wt.  of  UuO 

12.00 

12.00 

Glucose 

c.c.  NapSpOct 
Wt.  of  GuO 

14.52 

14.82 

12.45 

12.70 

Glucose  and  Aspara^in 

c.c.  NapSpQ^ 

14.54 

14.67 

Wt.  of  CuO 

12.47 

12.55 

Sucrose 

c.c.  NapS.^O^  . 

1.48 

1.23 

Wt.  of  Cu6 

1.27 

1.05 

Sucrose  and  Aspara.qin 

c«c*  ITspSpO/2 
Wt*  of  CuO 

2.93 

2.50 

2.55 

2.12 

Asparagin 

c.c.  lTapS.50„ 

i42 

.46 

Wt.  of  CuO  ^ 

.35 

.38 

Polarizations 

Sucrose,  5;a,  20*^0.,  400  ram. 

tube 

38.1° 

Asparagin  2/b(M40H  soln. ) 28°G.  , 400-mm. 

tiibe-  - 2.5° 

Glucose  lO/o,  280Q.,  400-nm 

tube 

60. 90 

Sucrose  90  c.c.,  Aspar.  10 

c .c . , 28^0 . , 

400  mm  33.6° 

Glucose  and  Aspar.  (spoiled 

by  bacterial 

action) 

Invert  Sugar  plus  10  c.c.  water,  £8°C.,  400-mm  -4.0 

Invert  Sugar  ’’  10  c.c.  Aspar.  ’’  *'  -2.2 


-19- 


The  results  of  j^xperiment  II  are  very  little  value  because  of 
insufficient  blanlc  determinations  to  enable  one  to  come  to 
definite  conclusions. 


jj^xperiment  III  Copper  Reduction 


Blanlc  s 

c • c • NapCpOa 
Wt.  of  CuO 


I 

.40 

.34£ 


II 

.58 

.356 


Amiuonia  solution  containg  15  c,c. 
in  100  c.c.  as  used  for 
Asparagine  solvent 


clc.  NapSpOij; 
Jt.  of  CuO 

.£5 

.£15 

.£3 

.197 

Invert  Su*R;ar 

c.c.  NagSgOg 
2^t.  of  GuO 

14.98 

1£.85 

III* 
(16. £4) 
(13.90) 

14.90) 

1£.78 

IV* 

(16.10) 
( 13.80) 

Invert  Sugar  and  ammonia 

c.c.  NapSpOa 
St.  of  CuO 

15.00 

1£.87 

III* 

( 15.38) 
( 13.19 

15.15 

1£.95 

IV^ 

(15.75) 

(13.49) 

Asparagin  and  ammonia 

c.c.  NapSoO„ 

Wt.  of  CuO 

£.5£ 

£.£6 

£.55 

£.£8 

Invert  Sugar,  Asparagin 
and  ammonia 

16.40 

14.0)5 

(15.87) 
( 13.60) 

13.45 

11.50 

( 13.18) 
(11. £9) 

* Samples  III  and  were  run  three  days  after  I and  II.  No 
reason  for  the  increase  in  reducing  power  has  suggested  itself. 

In  this  run  a determination  of  the  reducing  povi/er  in  the 
presence  of  ammonia  was  made  because  it  was  thought  that  the 
ammonia  toight  possibly  have  a polmerizing  effect  on  the  sugars 
which  would  effect  their  reducing  power.  The  very  poor  checks 
obtained  in  the  case  of  the  invertsugar,  asparagin  and  ammonia 
mixture  make  it  impossible  to  draw  any  conclusions  from  this 
experiment. 


I 


^ t * 


vex. 


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; ‘.X^ 


I . • f ‘t 

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V . ,i  C'‘.;  , 'f 


I?!  :, 


■-  *1?:. 


I *.• 


,:i  ■' 


f fr.v..  >.,  > 


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\.  '*h  S0  ii'i  if  »j 


tl  - A*  *:  . 

, ,(V- 


-20- 


■^jjeriment  III  continued 

iPolarizations 

Invert  Sugar,  26®  0.,  400-mra.  tube  Q -10-2®  V- 

Asparagin  in  ammonia,  26®  C-,  400-mra.  tube - 8.0®  V. 

Invert  Sugar  and  Asparagin,  26®  G.,  400-mm.  tube 

(equal  amounts)  - 9.2®  V. 


These  results  seem  to  indicate  that  the  effect  of  the 
asparagin  on  the  polarization  of  the  invert  sugar  is  additive 
only,  and  does  not  form  any  combination  v/ith  the  sugar 
changing  the  polarization  value  enough  to  explain  the  wide 
differences  obtained  by  Davis  and  other  wrrhers  v/ith  plant 
extracts- 


Dxperiment  III 


In  this  experiment  the  asparagin  was  dissolved  in  sodium 


carbonate  rather  than  ammonia.  The  excess  alkali  was  titrated 
Vvlth  ZQ'-/o  acetic  acid  till  the  solution  was  just  pink  to  phenol- 
phthalein. 

Invert  Sugar  and  Asparagin 


I 

II 

III 

NapSpO-z  c.c. 

9.05 

9.68 

9.74 

V7t.  of  GuO 

7.75 

8.30  • 

8.35 

Invert  Sugar  and  Asparagin 


Na„S^0„  c.c. 

9.26 

9.60 

9.19 

Yt.  bf^GuO 

7.92 

8.22 

7.82 

Polarizations 

Asparagin  20®  G. , 400-mm  tube .4®  Y. 

Invert  Sugar,  &iid  as^AragiA’  -2.90  v. 

Invert  Sugar  *3.00  V. 


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OONGLUSION 

Based  upon  the  data  obtained  in  these  experiments  the 
effect  of  the  asparagin  upon  the  reducing  power  and  polarization 
values  seems  to  additive  only,  and  is  not  the  chief  cause  for 
the  large  variations  in  the  sucrose  determinations  by  the  polar- 
iscopic  and  chemical  methods. 

SULuiARY 

1.  Wide  variation  between  the  sucrose  values  obtained  for  plant 
extracts  have  been  observed  by  many  chemists. 

2.  Somers  eliminated  the  possibility  that  the  variation  was 
due  to  the  effect  of  neutral  salts  in  solution,  or  to  auto-reduc- 
tion of  the  ffehlings  solution. 

3.  Davis  and  his  co-worhers  attributed  the  variation  to  the 
presence  of  amino  compounds  such  as  asparagin  and  glutamine,  but 
assuming  that  the  proper  neutrality  of  the  solution  was  maintainec 
this  seemed  improbable  unless  the  amino  substances  entered  into 
combination  with  the  sugars. 

4.  2taneh  and  I-Iaillard  prepared  some  compounds  by  the  reaction 
of  sugars  and  amino  substances  by  means  of  high  temperature  and 
pressure. 

5.  Sucrose  and  Glucose  were  purified  for  use  in  the  experimental 
work  of  the  problem. 

6.  A sugar  solution  was  analyzed  by  the  Defren’s,  Ifunson-Wallcer , 
and  polariscopic  methods  dmd  the  results  compared. 

7.  A modification  of  Low’s  iodometric  method  for  the  determin- 
ation of  copper  was  used  to  determine  the  copper  reduced. 


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-22- 


8.  The  effect  of  asparagin  upon  the  copper  reducing  power, 
and  rotatory  value  of  sucrose,  glucose  and  invert  sugar  was 
studied  v/ith  the  asparagin  in  ammonia  solution  and  in  sodium 
carbonate  solution  and  found  to  be  of  an  additive  nature  and 
insufficient  to  account  for  the  large  variations  which  are 
observed  between  the  sucrose  values  obtained  by  the  chemical 
and  polariscApiciiliethods* 


